Methods For Fermentation of Xylose and Hexose Sugars

ABSTRACT

Methods and systems for the isomerization and fermentation of xylose and hexose sugars using an immobilized enzyme system capable of sustaining two different pH microenvironments in a single vessel are disclosed. Bilayer particles are dispersed in a mixture comprising an ionic borate source and xylose. The bilayer particles have a first region with a first enzymatic activity comprising xylose isomerase and a pH of 6 or above, and a second region having a second enzymatic activity at an acidic pH.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under 35 U.S.C. §111(a) as a divisionalapplication which claims priority under 35 U.S.C. §119, 35 U.S.C. §120,and the Patent Cooperation Treaty to: parent application U.S. Ser. No.12/811,288 filed under 35 U.S.C. §371 on Jul. 23, 2010, now U.S. Pat.No. 8,507,232 issued Aug. 13, 2013; which claims priority toPCT/US2009/030033 filed under the authority of the Patent CooperationTreaty on Jan. 2, 2009, published; which claims priority to U.S.Provisional Application Ser. No. 61/009,973 filed under 35 U.S.C.§111(b) on Jan. 4, 2008. The disclosures of all priority applicationsare incorporated herein by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention generally relates to fermentation methods toproduce fuel from xylose and hexose sugars.

BACKGROUND OF THE INVENTION

Ethanol is being hailed as the fuel of the future. Interest in theproduction of fuel ethanol from renewable sources has increasedsignificantly. In order for fuel ethanol production to become apractical reality, cheaper substrates and more efficient productionprocesses are needed [1, 2]. Biomass, which includes all plant and plantderived material, forms a potential renewable source of sugars that canbe fermented to produce fuel ethanol and a variety of other fuels andchemicals. In addition to the many benefits common to renewable energy,biomass is particularly attractive because it is currently the onlyrenewable sustainable energy source for liquid transportation fuel.

Lignocellulosic biomass is an attractive energy feed-stock because it isan abundant, domestic, renewable source that can be converted to liquidtransportation fuels (From Biomass to Biofuels: A Roadmap to the EnergyFuture; Office of Science, US Dept. of Energy, December 2005).

Lignocellulosic biomass consists of three major components: cellulose(˜40-50%), hemicellulose (˜25-35%), and lignin (˜15-20%) [3]. Of these,cellulose and hemicellulose constitute the polysaccharides that can behydrolyzed to sugars that could be fermented to ethanol. In biomass, themajority of cellulose is a crystalline polymer of glucose that isrelatively difficult to hydrolyze into its monomeric sugar residues.Hemicellulose is a short branched polymer of pentose and some hexosesugars that surround the cellulose fibrils and is much less organized[4]. The pentose sugars consist primarily of xylose and to a smallerextent arabinose, while the hexose sugars are usually galactose andmannose. Due to its relatively open structure, the hemicellulosefraction is easier to convert to its sugar monomers by variouspretreatment techniques than the cellulose fraction.

For the conversion of lignocellulosic biomass to bioethanol to beeconomically feasible, it is imperative that the hemicellulose-derivedmonomeric sugars be fermentable along with the glucose derived fromcellulose. Unfortunately, no known native (or wild type) microorganismsare able to efficiently ferment both glucose and xylose to ethanol. Wildtype Saccharomyces cerevisiae strains can readily ferment glucose aswell as other sugar components of biomass like mannose, fructose andgalactose [5]. Xylose, which forms a major portion of hemicellulose,cannot be fermented by the same native strains of yeast. Severalnon-Saccharomyces strains of yeast, such as Pichia stipitis and Candidashehatae, are known to ferment pentose sugars more efficiently thanother yeasts [6]. In such yeasts, the xylose metabolism pathway goesfrom xylose to xylitol to xylulose [7, 8]. In other yeast strains aswell as bacteria and fungi, xylose can also be converted to xylulose viaa single enzyme, xylose isomerase (XI). Several yeasts, including S.cerevisiae, that cannot ferment xylose are able to ferment xylulose, theketose isomer of xylose [9-12] to ethanol. Considerable effort has beenfocused on the genetic modification of microorganisms so that bothxylose and glucose can be efficiently metabolized using the sameorganism [13-25].

While genetically modified organisms (GMOs) have potential forfermentation of pentose and hexose sugars, their genetic stability,overall ethanol yield, and ability to survive under the conditions ofindustrial fermentation are unproven [26, 27]. Hence, an alternativeapproach to fermentation of xylose to ethanol involves using nativeyeast strains with the addition of exogenous enzymes for theisomerization of xylose. In this approach, the production of xylulose isaccomplished using immobilized glucose/xylose isomerase [11, 28-30]. Theappeal for this approach is that XI, along with amylase and protease, isamong the most widely and cheaply available commercial enzymes [31].Hydrolysate from lignocellulosic biomass will contain both xylose andglucose. The affinity of XI for xylose is typically 1 to 2 orders ofmagnitude greater than its affinity for glucose; hence, isomerization ofxylose to xylulose will dominate over isomerization of glucose tofructose [31]. However, any fructose formed is readily fermentable bySaccharomyces to produce ethanol, so fructose formation is not a causefor concern.

Although XI is capable of converting xylose to xylulose, underconditions where XI has significant activity, the equilibrium ratio ofxylose:xylulose is typically high (on the order of 5:1) [32-34]. Hence,xylose isomerization does not have a favorable forward equilibrium. Oneway to increase xylose conversion is to drive the isomerization forwardby removal of the product xylulose. Simultaneous isomerization andfermentation (SIF), where the isomerization of xylose and thefermentation of xylulose to ethanol occur simultaneously in the samevessel, is one method for increasing xylose utilization. However, SIFdoes have inherent limitations due to the pH range over which XI isactive. All commercially available XI's have optimal activity at pH 7 to8, and the XI activity drops sharply as the pH decreases. In contrast,the optimal pH for the fermentation is in the range of 4 to 5. The largepH difference associated with these two steps poses a problem forconducting SIF efficiently. The SIF can be carried out at a compromisedpH between 4 and 7, but the results are less than optimal for bothreactions [11]. Efforts to isolate a XI with optimal activity atsignificantly lower pH for SIF were also noted in the literature [30].However, it does not appear that this enzyme has the same level ofactivity as displayed by the commercially available enzymes.

The instant invention provides a further improvement over one of theco-inventor's prior inventions disclosed in the Fournier et al. U.S.Pat. No. 5,254,468 and the Fournier et al. U.S. Pat. No. 5,397,700, thedisclosures of each of which are incorporated herein by reference intheir entireties.

Considering the above-mentioned concerns, it is clear that there remainsa need in the art for a method of developing a process that enablesefficient fermentation of xylose and hexose sugars.

SUMMARY OF THE INVENTION

In a first broad aspect, there is provided a method of fermenting one ormore sugars including xylose, comprising dispersing particles in amixture comprising a borate source and xylose, the particles includingone or more co-immobilized enzymes, and fermenting the mixture. The useof bilayer particles comprising an inner core having a first enzymaticactivity and an outer region having a second enzymatic activity isfurther contemplated. In an embodiment of the invention, it iscontemplated that the bilayer particles comprise xylose isomerase (XI)and urease.

In one embodiment of the invention is provided a method of improving afermentation process comprising, or alternatively consisting orconsisting essentially of, the following steps: i) dispersing one ormore co-immobilized enzyme particles into a mixture containing a borateadditive and xylose, the co-immobilized particles having an outer layercomprising urease and an inner core comprising xylose isomerase (XI) (orboth of the enzymes) co-immobilized; ii) diffusing xylose from themixture to the inner core where XI is active and xylulose formed; iii)diffusing at least a quantity of the boron, in an ionic form, from theborate additive into the inner core of, at least, some particles; iv)reacting the ionized borate with the xylulose to form an ionizedborate-xylulose complex; v) migrating the ionized borate-xylulosecomplex to the mixture; and (vi) dissociating the ionizedborate-xylulose complex in the mixture to obtain free xylulose. It is tobe understood that some steps of this process may occur passively and donot require an affirmative step.

In certain embodiments, wherein the mixture has an acidic pH and atleast a portion of the pellet has a different pH. Also, in certainembodiments, the pH at least a portion of the pellet is about 7 to about8 and the pH of the mixture is about 4 to 5.5.

In certain embodiments, the mixture has an acidic pH, and an inner coreof the pellet has a different pH. In certain embodiments, the pH of theinner core is about 7 to about 8 and the pH of the mixture is about 4 toabout 5.5.

In certain embodiments, the borate additive comprises, or alternativelyconsists of, sodium tetraborate.

In certain embodiments, the ionized borate comprises, or alternativelyconsists of, tetrahydroxyborate ion.

In another broad aspect, there is provided a method of fermenting one ormore sugars including xylose, comprising dispersing bilayer pellets in amixture containing urea, a borate source, and a substrate, wherein thebilayer pellets comprise a porous outer region including immobilizedurease and a porous inner core including an immobilized enzyme otherthan urease that acts on the substrate, and fermenting the mixture.

In another embodiment there is provided a method of producing a productusing co-immobilized bilayer pellets having an outer layer of a porousmaterial containing immobilized urease and an inner core of a porousmaterial containing an immobilized enzyme other than urease that acts ona substrate to produce the product, the method comprising, oralternatively consisting of or consisting essentially of, the followingsteps: i) dispersing the bilayer pellets in a mixture containing urea, aborate additive, and the substrate, the mixture having an acidic pH; ii)reacting the urease with urea diffusing into the outer layer to produceammonia, which consumes hydrogen ions diffusing toward the inner core toprovide an inner core with a pH higher than the acidic pH of themixture; iii) reacting the immobilized enzyme in the inner core with thesubstrate as it diffuses into the inner core to produce the product; iv)reacting the ionized borate with the product to produce an ionizedborate-product complex; and v) migrating the ionized borate-productcomplex to the mixture, where it dissociates to release free xylulose.It is to be understood that some steps of this method may occurpassively and do not require an affirmative step.

In one embodiment of this method, the immobilized enzyme in the innercore is xylose isomerase and the product is xylulose.

In another broad aspect, there is provided a method for the simultaneousisomerization and fermentation (SIF) of xylose to ethanol comprising, oralternatively consisting of or consisting essentially of, the followingsteps: i) immobilizing xylose isomerase in a porous polymer material soas to form a substantially spherical particle, the particle forming aninner core region of a larger pellet; ii) mixing the particles with atleast water, urease, a monomer, a crosslinking agent, and apolymerization initiator so as to form an aqueous medium, the aqueousmedium and particles comprising an aqueous suspension; iii) maintainingthe aqueous suspension at a temperature between about 0° C. to about 4°C.; iv) adding at least toluene, chloroform, and a surfactant to thesuspension so as to form an aqueous hydrophobic phase; v) agitating thehydrophobic phase under nitrogen conditions and at a temperature betweenabout 0° C. to about 4° C. to allow polymerization of the monomer and toform a thin polymer coating containing the urease immobilized thereinaround the particles to form bilayered immobilized enzyme pellets; vi)mixing at least xylose feedstock, urea, and yeast cells having highethanol productivity and ethanol tolerance so as to form a bulk liquid,the bulk liquid being placed in a closed reactor having agitation means,vii) setting and adjusting the pH of the bulk liquid so as to maintainthe pH in the range of about 4.0 to about 5.5, viii) dispersing thebilayered immobilized enzyme pellets in the bulk liquid; ix) adding aborate additive to the bulk solution; x) diffusing xylose into the innercore region of the pellet; xi) isomerizing the diffused xylose toxylulose by contact with xylose isomerase immobilized in the inner core;xii) diffusing the xylulose out into the bulk liquid; xiii) providing apH in the inner core region of about 7.0 to about 8.0 by diffusing ureainto the outer layer of the pellet, whereby the urea is hydrolyzed toammonia by the immobilized urease, the ammonia neutralizing hydrogen orpositively charged ions that diffuse from the bulk liquid into the innercore region of the pellet; xiv) agitating the bilayered pellets and bulkliquid under substantially anaerobic conditions and at a temperature andfor a sufficiently long period of time so as to allow the fermentationof xylulose to ethanol; and, xv) fermenting xylulose to ethanol whichoccurs substantially contemporaneously with the isomerization of xyloseto xylulose. It is to be understood that some steps of this method mayoccur passively and do not require an affirmative step.

It is further to be understood that thesimultaneous-isomerization-and-fermentation method described hereinworks with: i) porous monolayer particles wherein the enzymes xyloseisomerase and urease are co-immobilized together; and ii) bilayerparticles that have inner and outer layers, wherein the inner layercontains xylose isomerase and the outer layer contains urease. It isalso to be understood that the term “co-immobilized” herein can refer toboth embodiments. It is further to be understood that the termsparticles and pellets can be used interchangeably.

In another embodiment, there is provided method for the simultaneousisomerization and fermentation of xylose to ethanol, comprising thesteps of mixing xylose, urea, and yeast cells having high ethanolproductivity and ethanol tolerance in a reactor to form a liquidmixture, maintaining the pH of the mixture in the range of about 4.0 toabout 5.5, dispersing co-immobilized enzyme particles having at leasttwo enzymes in the mixture, adding a borate source to the mixture,diffusing xylose into the particles, isomerizing the diffused xylose toxylulose by activity of immobilized xylose isomerase, diffusing thexylulose out into the mixture, maintaining the pH of the liquid in theparticle during isomerization in the range of about 7.0 to about 8.0 bydiffusing urea into the particle, whereby the urea is hydrolyzed toammonia by immobilized urease, the ammonia neutralizing hydrogen ionsthat diffuse into the pellet, agitating the mixture under substantiallyanaerobic conditions so as to allow the fermentation of xylulose toethanol, and fermenting xylulose to ethanol.

The use of bilayer particles comprising an inner core having a firstenzymatic activity and an outer region having a second enzymaticactivity is further contemplated. In an embodiment of the invention, itis contemplated that the bilayer particles comprise xylose isomerase(XI) and urease.

In another embodiment, the fermentation is conducted in a closed reactor(or fermentor) with agitation.

In certain embodiments, the particles can have a polymer coating. Onenon-limiting example of such coating is polyacrylamide.

In certain embodiments, the borate additive comprises, or alternativelyconsists of, sodium tetraborate.

In certain embodiments, the method includes one or more of thefollowing: step ix_(a)) reacting the borate additive to produce anacid-containing boron; step x_(a)) diffusing the boron-containing acidinto the pellets and reacting the xylulose with the boron-containingacid to produce a complex of xylulose and borate ion; and step xii_(a))diffusing the xylulose and borate ion compound into the bulk liquid.

In certain embodiments, the boron-containing acid comprises, oralternatively consists of, boric acid. In certain embodiments, theborate ion comprises, or alternatively consists of, tetrahydroxyborateion.

Further contemplated is a reaction medium comprising xylose, a boratesource, and urea.

In another embodiment of the invention, the methods described hereinprovide high yields of xylulose from xylose isomerization and highpercentages of xylulose and glucose conversion into ethanol using nativeS. cerevisiae.

The invention provides fermentation methods for enhancing conversion ofxylose to xylulose, increasing the rate of production of ethanol, andreducing the overall time required for fermentation of both C6 and C5sugars.

In yet another embodiment, there is provided a borate-enhancedisomerization of xylose in a co-immobilized enzyme pellet systemcomprising a two-pH environment system. In certain embodiments, thesystem includes adding borax (sodium tetraborate). In certainembodiments, the two-pH environment system includes induction of apositive shift in the xylose:xylulose equilibrium resulting from aselective complexation of xylulose to tetrahydroxyborate ions formedfrom borax. In certain embodiments, the system includes a substantiallysimultaneous isomerization and fermentation (SIF) step capable ofsustaining two different pH-microenvironments in a single vessel,wherein the first pH is substantially optimal for xylose isomerization,and the second pH is substantially optimal for fermentation of xylulose.In certain embodiments, the SIF step includes co-immobilization ofurease with xylose isomerase.

In another broad aspect, there is provided herein a novel configurationinvolving a packed bed of co-immobilized enzyme pellets that isconnected in series to a porous hollow fiber membrane fermentor (HFMF).The packed bed configuration provides a fermentation beer that is freefrom yeast and can be easily concentrated for ethanol recovery. Inaddition, after the ethanol has been distilled off from the fermentationbeer, the remaining aqueous solution containing buffers and borate canbe recycled to upstream process units (i.e., hydrolysis/isomerization),resulting in significant cost savings in consumables. The packed bedconfiguration also provides a facile method for recovery and reuse ofthe isomerization catalyst pellets since the pellets are confined to thepacked bed and do not come into direct contact with yeast. The packedbed configuration allows for a high density of yeast in the HFMF whichis needed for xylulose fermentation and also allows extended use of theyeast for fermentation. Unlike traditional fermentors, the yeast is notdisposed of after each batch of fermentation. The modular nature of thepacked bed configuration allows for easy scale-up of the SIF processwithout significant capital costs.

Various objects and advantages of this invention will become furtherapparent to those skilled in the art from the following detaileddescription of the preferred embodiment(s) and the examples, when readin light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Cross-section of immobilized XI, e.g. SWEETZYME™ pellet showingthe steady-state pH profile developed when urease is co-immobilized inthe pellet and urea is added to the fermentation broth. The pH in thefermentation broth is pH₀, which is typically in the range of 4 to 5.Zone 1 (outer layer) of the pellet contains immobilized urease andrepresents the region of the pellet where the pH changes with radialposition as ammonia is produced by the consumption of urea. Zone 2(core) represents the region of the pellet which is at pH₂, the elevatedpH. The boundary between zones 1 and 2 represents either the point whereall urea is consumed or the penetration depth of urease into the pellet.

FIG. 2: Graph showing that two pH microenvironments are developed in theco-immobilized enzyme system via urea hydrolysis. Solid symbols are usedfor unaltered SWEETZYME™; open symbols are used for the XI/ureaseco-immobilized enzyme pellets. The three experiments shown are: (A) pH7.5; (B) pH 4.5 with 0.01M urea, and (C) pH 4.5 with no urea; each used0.13 g pellets. Unaltered SWEETZYME™ yielded no xylulose production atpH 4.5 (data not shown). Xylulose production shown for B indicates thatXI has activity when urea is added. The initial xylose concentration is60 g/l.

FIG. 3: Graph showing borate favorably shifts the xylose/xyluloseequilibrium for both unaltered and co-immobilized enzyme pellets. Solidsymbols are used for unaltered SWEETZYME™; open symbols are used for theXI/urease co-immobilized enzyme pellets. The three experiments shownare: (A) pH 7.5; (B) pH 4.5 with 0.01M urea, and (C) pH 7.5 with nourea. All three experiments show a significant shift in the equilibriumtoward xylulose production. Only the conditions represented by curve Bare conducive for simultaneous isomerization and fermentation. Theinitial xylose concentration is 60 g/l.

FIG. 4: Schematic illustration showing role of xylulose-boratecomplexation in the co-immobilized enzyme system. When sodiumtetraborate (borax) is added to solution, it dissociates intotetrahydroxyborate (borate, B) ion and boric acid. In the pelletinterior, higher pH favors tighter xylulose-borate binding (Xu-B complexformation), which effectively reduces the xylulose concentration in theinterior and forces the isomerization forward. In the bulk, the lower pHhas an uncoupling effect on the Xu-B complex, making the dissociatedxylulose readily available to the yeast. Removal of xylulose viafermentation further forces dissociation of the xylulose-borate complex.Dashed lines represent transport of species; solid lines representreactions.

FIG. 5: Graph showing the effect of XI/urease activity on theisomerization kinetics and xylose/xylulose production for theco-immobilized enzyme pellets. All pellets were from the sameco-immobilization batch and have the same urease and XI activities per gpellet at pH 7.5. The initial urea concentration used in all experimentswas 0.01M. The improvement in the xylulose yields with increased enzymeloading can be attributed to the dual role of tetrahydroxyborate ions inthe co-immobilized enzyme pellet system. The initial xyloseconcentration is 60 g/l.

FIG. 6: Graph showing effect of initial urea concentration on theisomerization kinetics and xylulose production for the co-immobilizedenzyme pellets. Both experiments use 0.13 g pellets from the sameco-immobilization batch and have the same urease and XI activities per gpellet at pH 7.5. The decrease in the rate of isomerization and xyluloseproduction seen in curve A is due to consumption of urea. In curve B,the urea concentration is high enough that the rate of xyluloseisomerization does not appear to be affected by urea consumption overthe entire 48 hr period. The initial xylose concentration is 60 g/l.

FIG. 7: Graph showing the effect of initial urea concentration and massof pellets on xylulose production for the co-immobilized enzyme pellets.White bars are xylose; hatched bars are xylulose. All pellets (A-E) werefrom the same co-immobilization batch and have the same urease and XIactivities per g pellet; initial pH was 4.5. Results for unalteredSWEETZYME™ are given in F. All experiments contained 0.05M borate in 25ml of broth. The percentage of total xylulose is given above the barsfor each experiment. The time of apparent equilibrium and mass ofpellets used is indicated on the x axis.

FIG. 8: Graph showing that the addition of metal ions results in a smallshift in the isomerization toward xylulose, but the effect is not assignificant as the shift associated with addition of sodium tetraborate.If added, sodium tetraborate was 0.05M and metal ions were 20 mM MgCl₂and 1 mM CoCl₂. All data shown are for unaltered SWEETZYME™ at pH 7.5.The four experiments shown differ by additives and are: (A) noadditives; (B) metal ions; (C) borate; and (D) borate and metal ions.

FIG. 9: Flow chart showing a packed bed and fermentor moduleconfigurations for a simultaneous isomerization and fermentation “SIF”process. The hollow fiber membrane fermentor (HFMF) shows a blow-up of amicroporous fiber and yeast cells. Sugar flows through fiber lumens andyeast grow in the space surrounding the fibers. Sugars diffuse throughpores in the fiber wall to yeast, where sugars are fermented to ethanol.Ethanol diffuses back through the pores into the fiber lumen and flowsout of the device at the end. The yeast cells are confined to the shellspace and can be loaded at a very high density per unit volume as shown.

FIG. 10: Isomerization kinetics for the co-immobilized enzyme pellets inthe packed bed and shake flask. All pellets were from the sameco-immobilization batch and have the same urease and XI activities per gpellet at pH 7.5. The initial urea concentration used in all experimentswas 0.05M. Isomerization kinetics are significantly faster for thepacked bed experiment as compared to the control experiment in theagitated shake flask.

FIG. 11: Results for glucose fermentation in the HFMF. The initialglucose concentration is 60 g/l. 25 g of yeast cells were packed intothe extracapillary space of the HFMF. Fermentation was conducted usingdifferent media flow rates for 24 hours. The ethanol production ratepeaked at a flow of 30-50 ml/min with nearly 100% ethanol yield. Thetheoretical ethanol yield is 0.51 g ethanol/g glucose.

FIG. 12: Spinning basket catalytic pellet confinement that is submergedin a fermentor.

FIG. 13: Examples of process configurations using co-immobilized enzymetechnology. Process A—Using co-immobilized enzyme pellets withcellulases at pH 4.8 and 50° C. in the presence of borate and urea canenable simultaneous saccharification and xylose isomerization (SSI). Theresulting sugar mix of glucose and xylulose can be fermented by nativeyeasts. Process B—Adding co-immobilized enzyme pellets and native yeastto biomass hydrolysate at pH 4.5 and 35° C. in the presence of borateand urea allows simultaneous isomerization and fermentation (SIF) withclose to theoretical ethanol yields from all biomass sugars. ProcessC—Simultaneous saccharification and fermentation (SSIF) of C6 and C5sugars using native yeasts can be done with co-immobilized enzymepellets in a medium maintained at pH 4.8 and 35° C. containing borate,urea, cellulases, and native yeast.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, genera, and reagentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. All technicaland scientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs unless clearly indicated otherwise.

Of the sugars recovered from lignocellulose, D-glucose can be readilyconverted into ethanol by baker's or brewer's yeast (Saccharomycescerevisiae). However, xylose that is obtained by the hydrolysis of thehemicellulosic portion is not fermentable by the same species of yeasts.Xylose fermentation by native yeasts can be achieved via isomerizationof xylose to its ketose isomer, xylulose. Isomerization with exogenousxylose isomerase (XI) occurs optimally at a pH of 7-8 while subsequentfermentation of xylulose to ethanol occurs at a pH of 4-5.

Methods useful for achieving isomerization and fermentation aredescribed throughout the instant application, including those methodsand processes set forth previously in the Summary of the Invention.

In a first broad aspect, there is provided a method for the efficientisomerization of xylose to xylulose by using an immobilized enzymesystem capable of sustaining two different pH microenvironments in asingle vessel, the systems also providing conditions suitable for thefermentation.

A two-enzyme pellet test of the method described herein shows conversionof xylose to xylulose even when the immobilized enzyme pellets aresuspended in a bulk solution whose pH is sub-optimal for XI activity.The co-immobilized enzyme pellets can be useful in effectivelyconducting “simultaneous isomerization and fermentation” (SIF) ofxylose.

In a particular embodiment, to help further shift the equilibrium infavor of xylulose formation, sodium tetraborate (borax) was added to theisomerization solution. Binding of tetrahydroxyborate ions to xyluloseeffectively reduces the concentration of xylulose and leads to increasedxylose isomerization.

In another particular embodiment, the addition of 0.05M borax to theisomerization solution containing such co-immobilized enzyme pelletsresulted in xylose to xylulose conversion as high as 86% under pHconditions that are suboptimal for XI activity. As such, the methoddescribed herein is adaptable for industrial conditions and providessignificant increases in the yield of ethanol from xylose in an SIFapproach.

To overcome the disparity in the optimal pH's for the isomerization andfermentation, there is now described a novel method of isomerizationthat incorporates urease co-immobilized with xylose isomerase. Thismethod uses co-immobilized enzyme pellets comprising XI immobilized in aporous pellet for isomerization and the immobilized urease enzyme for pHcontrol (FIG. 1).

When the co-immobilized enzyme pellets are dispersed in a fermentationbroth which contains urea in addition to the other necessary ingredientsfor fermentation, it is possible to sustain a significant pH gradientbetween the bulk liquid and the core region of the pellet because ashydrogen ions diffuse into the pellet, they are neutralized by theammonia produced in the hydrolysis of urea by urease. The XI, which ismaintained at a higher pH in the inner core of the pellet, thencatalyzes the isomerization of the xylose to xylulose; xylulose diffusesfrom the pellet and is then available for fermentation in the bulksolution.

Preferably, the concentration of urea used in the fermentation broth isbetween about 0.01 M urea and about 0.1 M urea. In a particularlypreferred embodiment of the invention, the urea concentration is about0.1 M urea.

Although the co-immobilized enzyme method is able to sustain thenecessary pH difference between isomerization and fermentation steps inSIF, the overall production rate of ethanol in SIF, may be limited bythe total concentration of xylulose available to the yeast. Under normalequilibrium conditions, the xylulose concentration is usually at bestone fifth of the xylose concentration. Hence, there is a need for amethod of shifting the equilibrium towards higher xylulose formationthat will further increase the rate of ethanol production.

The method further includes the use of a borate additive in thefermentation broth, which provides a shifting of the xylose:xyluloseequilibrium towards increased xylulose formation. In one embodiment ofthe invention, the borate additive is sodium tetraborate. In anon-limiting hypothesis of the invention, it is believe that the borateion may shift the equilibrium between xylose and xylulose in XIcatalyzed isomerization from about 20:80 to about 70:30. It is believedthat the borate ion binds more tightly to xylulose than xylose,effectively reducing the product concentration, and thus shifting theequilibrium toward increased xylulose formation. This ability of borateto bind to xylulose is pH dependent, with higher pH (6 to 7.5) favoringtighter binding. Thus, as the pH increases, the concentration of freexylulose decreases. Therefore, the rate of fermentation of xylulose inthe presence of borate is also pH dependent, with lower pH leading tohigher free xylulose concentrations and thus higher yields and rates ofethanol production.

In certain co-immobilized enzyme methods that provide differentmicroenvironments for isomerization and fermentation, themicroenvironments can benefit by the addition of borate to thefermentation broth. Inside the co-immobilized enzyme pellet, the pH iselevated, XI is active, and the isomerization equilibrium is favored bystrong borate binding to xylulose. In contrast, in the low pHfermentation broth, borate has a reduced binding affinity for xylulose,and thus produces a higher free xylulose concentration for fermentationto ethanol.

The co-immobilized enzyme method described herein is particularlyeffective for isomerization in conjunction with conditions optimal forfermentation by common S. cerevisiae. Further producers of ethanol fromxylulose and glucose that are useful with the invention include, but arenot limited to, species of Brettanomyces, Schizosaccharomyces,Torulaspora, Saccharomyces, Pachysolen, Kluyveromyces, and Hansenula.

Conventional fermentation techniques, which are well known in the art,may be used to ferment the xylulose and glucose sugars in the bulkliquid into ethanol. As is typical in the fermentation process, largelyanaerobic conditions and fermentation temperatures maintained betweenabout 30° C. and 40° C. are useful for performing the methods andprocesses set forth herein, although it is to be understood thatmicroaerophilic conditions may also be useful.

The glucose and xylose sugars may be derived from lignocellulosebiomasses by conventional techniques for the hydrolysis oflignocellulose. Cellulose, which provides glucose feedstock, isdifficult to hydrolyze due to its crystalline structure and closeassociation with lignin in the biomass. In contrast, the amorphousstructure of hemicellulose allows it to be easily hydrolyzed by a weakacid into its constituent sugars, namely xylose as well as arabinose andglucose.

In a particular embodiment, the media composition both shifts theequilibrium in favor of xylulose production and improves XI activity.Also, in certain embodiments, borate and/or other metal ions are usefulto modulate the kinetics and/or equilibrium of the isomerizationreactions. Thus, in certain embodiments, divalent metal ions are usefulto increase the long-term activity of XI. In a preferred embodiment ofthe invention, the divalent metal ions are selected from magnesium(Mg²⁺) and cobalt (Co²⁺).

These advantages will now be illustrated by the following non-limitingexamples. The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions. Allpublications, including patents and non-patent literature, referred toin this specification are expressly incorporated by reference. Thefollowing examples are intended to illustrate certain preferredembodiments of the invention and should not be interpreted to limit thescope of the invention as defined in the claims, unless so specified.

EXAMPLES

Materials and Methods:

Chemicals:

Novo SWEETZYME™ (Sigma Aldrich G4166, ≧350 U/g with activity based onisomerization of glucose to fructose), which is immobilized glucoseisomerase produced from Streptomyces murinus, and Genencor GENSWEET™ IGI(220 U/g with activity based on isomerization of glucose to fructose),which is immobilized glucose isomerase produced from Streptomycesrubiginosus was used for the isomerization of xylose. The immobilizedglucose isomerase has optimal activity for glucose/fructoseisomerization at pH 7.5-7.8 and 54-60° C. (as per the manufacturer). TheSWEETZYME™ or GENSWEET™ pellets were dry, brown, cylinder-shapedgranules with a diameter of approximately 1-3 mm Jack bean urease (SigmaU4002, 70,400 U/g) was used for generating the co-immobilized enzymepellets used in the isomerization studies. Urease has optimal activityat pH 7.0 and 25° C. (as per manufacturer). Both enzymes were stored at4° C. Additional chemicals, including xylose, urea, borax, magnesiumchloride, cobalt chloride, sodium citrate, and Tris were all purchasedfrom Sigma Aldrich (St. Louis, Mo.).

Immobilization of Urease on SWEETZYME™ and GENSWEET™ Pellets:

For co-immobilization of urease on the pellets, 500 ml of 1 g/l or 2 g/lurease solution and 2 g of SWEETZYME pellets were added to a 1 literbeaker. The beaker was left on the benchtop at room temperature for 24or 48 hrs to form co-immobilized enzyme pellets.

The co-immobilized enzyme pellets were separated from the solution bydecanting and gravity filtration and dried on a paper towel at roomtemperature for 24 hrs or until dry. The co-immobilized enzyme pelletswere stored at 4° C. until use. Activity of immobilized urease wasmeasured at pH 7.5 and 25° C. using a standard assay procedure thatmeasures the rate of ammonia liberation. The urease activities obtainedwith this immobilization procedure were in the range of 550-577 U/gpellets, where a Unit liberates 1 μmol of ammonia per minute under theassay conditions.

Measurement of Xylose Isomerization Kinetics and Equilibrium:

All experiments were carried out at 34° C. in a volume of 25 ml in 50 mlshake flasks agitated at 130 rpm in an incubated shaker. Each experimentwas conducted in duplicate. All experiments used 60 g/l xylose, andunless otherwise noted, 5.2 g/l of enzyme pellets (0.13 g) was used foreach experiment. Buffered solutions used in making the isomerizationmedia were 0.01 M Tris buffer (pHed to 7.5 using 0.01 M NaOH) and 0.05 Msodium citrate buffer (pHed to 4.5 using citric acid). In experimentswith co-immobilized enzyme pellets, urea concentration was 0, 0.01 or0.1 M.

Analytical Techniques and Data Analysis:

To analyze experiments for xylose and xylulose concentration, a 200 μlsample was collected at each time point. The sample was diluted 1:3 withdeionized water and then filtered through a 0.2 μm filter. Xylose andxylulose calibration standards with concentrations ranging from 0.25 to80 g/l in pH 4.5 citrate buffer were prepared in a similar manner. Allstandards and samples were analyzed by HPLC using a 30 μl injectionvolume with a 100 μl injection loop. The HPLC unit used was a ShimadzuSeries 10A HPLC unit equipped with a SIL-10Ai autosampler and arefractive index detector (RID 10A). A Bio-Rad Aminex HPX-87 P (300×7 8mm) ion exchange column was used for sugar analysis using a mobile phaseof deionized water with a flow rate 0.6 ml/min and a temperature of 80°C. This column was successful in separating xylose and xylulose. Todetermine if the xylulose-borate complex dissociated and elutedseparately, solutions of borate and borate with xylulose were injected,and the area of the borate peak was measured. Since the height of theborate peak was independent of xylulose concentration, the inventors nowbelieve that the xylulose-borate complex dissociated into xylulose andborate, and the xylulose peak represented total xylulose in the mixture.Finally, data for xylose and xylulose concentration at each time pointwere summed and normalized to 60 g/l total concentration to eliminatevariability and to close the mass balance. All experiments wereperformed in duplicate and data were very reproducible; data shown isrepresentative of one run.

Results and Discussion:

The co-immobilized enzyme pellet system is able to achieve two differentpH microenvironments within a single vessel—one optimal for XI activityand the other suitable for conducting fermentation. In addition, thesodium tetraborate decahydrate (borax) is able to alter the kinetics andshift the xylose/xylulose equilibrium.

Sustainability of Two-pH Environments in a Single Vessel

Unaltered Pellets:

As an initial control experiment, the isomerization of xylose toxylulose was studied using SWEETZYME™ pellets, as received (i.e., withno co-immobilization with urease. The time course of xylose consumptionand xylulose formation was monitored in a 25 ml solution for an initialxylose concentration of 60 g/l with 0.13 g pellets at 34° C. Theisomerization mixture was buffered at pH 7.5, which is the optimal pHfor XI activity.

As seen in FIG. 2 curve A, the concentration of xylulose steadilyincreased and reached an equilibrium value of about 9 g/l, suggesting anequilibrium xylose:xylulose ratio of nearly 6:1 under these conditions.When the same experiment was repeated at a reduced pH of 4.5, noxylulose was detected in the reaction mixture, even after 40 hrs (datanot shown). At a pH of 4.5, XI is 3 pH units below its optimum, anddisplays essentially no activity.

XI/Urease co-immobilized enzyme pellets:

The co-immobilized enzyme pellets were formed by adsorbing urease ontothe SWEETZYME™ pellets.

The co-immobilized enzyme pellets (0.13 g) were added to 25 ml ofreaction media containing 60 g/l xylose buffered to pH 4.5. As with theunaltered pellets, no xylulose formation was observed under theseconditions even after 48 hrs (see FIG. 2, curve C). Next, 0.01M urea wasadded to the bulk solution buffered to a pH of 4.5. Formation ofxylulose was observed in the presence of urea, and the concentration ofxylulose in the reaction medium gradually increased to reach a value ofabout 5 g/l by 48 hours (see FIG. 2, curve B).

The production of ammonia by urea hydrolysis catalyzed by immobilizedurease in the pellets raises the internal pH within the core of thepellets, as shown in FIG. 1. In certain embodiments, the interior pHmust be well above the bulk pH of 4.5 in order for the XI within thepellets to be catalytically active. Therefore, when the xylose in thebulk solution diffuses into the pellets and reaches a higher pH regionwhere XI is active, xylose isomerizes to form xylulose. At the sametime, the continuous production of ammonia in the outer layer (Zone 1,FIG. 1) of the co-immobilized enzyme pellets also tends to neutralizeany hydrogen ions that diffuse into the core of the pellets from thebulk solution, thereby sustaining the pH difference between the interiorof the pellets and the external solution.

Because the rate of isomerization in curve B, shown in FIG. 2, is lowerthan that obtained at pH 7.5 in unaltered pellets, it shows that theinterior pH is not maintained at 7.5 but at a suboptimal pH, eitherabove or below 7.5. If the interior pH is suboptimal, then the XIactivity will be lower than that in the unaltered pellets at pH 7.5, andthe time required to reach equilibrium will be longer. If XI activity isreduced in the co-immobilized enzyme pellets, the time required forisomerization may ultimately exhaust the urea from the bulk solution, atwhich point XI activity will be lost, and the isomerization reactionwill cease.

The interior pellet pH is a function of the urease loading as well asthe urea concentration profile in the pellet. The Michael is constant(K_(m)) for urease hydrolysis of urea is 2.9 mM, so with 0.01 M (10 mM)urea, urea is initially being consumed at approximately 78% of V_(max)at the surface of the pellet. Increasing the bulk concentration of urearesults in increased ammonia production and an increase in the interiorpellet pH. Depending upon whether the interior pH is above or below thepH for optimum XI activity, an increase in interior pH will decrease orincrease the rate of xylose isomerization.

To achieve a desired isomerization in the co-immobilized enzyme pelletsystem, the urea concentration in the bulk solution can be optimized fora specific urease loading and can be maintained at a constantconcentration throughout the isomerization to allow maximal, constant XIactivity.

Significant XI activity in the co-immobilized enzyme pellets has beendemonstrated at a bulk pH of 4.5 with 0.01M urea. Since the overallproduction rate of ethanol is limited by the total concentration ofxylulose available to the yeast, in certain embodiments, it is desiredto modify conditions to favorably enhance the isomerization and thexylose:xylulose proportions. In certain embodiments, the addition ofborate to the reaction medium is used to enhance the isomerizationkinetics and to favorably shift the equilibrium.

Effect of Sodium Tetraborate Addition on Xylose Isomerization

Mechanism of Sugar-Borate Complexation:

Borate leads to a shift in the equilibrium isomerization due to thebinding of tetrahydroxyborate ions to aldose and ketose sugars. At nearneutral pH, tetrahydroxyborate ions can be formed by hydrolysis of borax(Na₂B₄O₅(OH)₄.8H₂O):

B₄O₅(OH)₄ ²⁻+5H₂O

3B(OH)₃+B(OH)₄ ⁻+OH⁻  (1)

The boric acid produced in the above reaction is a weak-acid (pK_(a)˜9)that ionizes to a slight extent by reaction with water at neutral pH toform additional tetrahydroxyborate ions:

B(OH)₃+H₂O

B(OH)₄ ⁻+H⁺  (2)

The tetrahydroxyborate ions produced in the above reactions are able tocomplex with adjacent hydroxyls on sugar molecules. As shown inEquations 3a and 3b, each tetrahydroxyborate ion can bind up to twomolecules of sugar in a two-step process.

Borate is able to complex, via the above mechanism, more readily withthe open-chain structure of xylulose as compared to the cyclichemiacetal form of xylose. This binding preference leads to a shift inthe xylose:xylulose isomerization equilibrium in favor of xyluloseformation.

Unaltered Pellets:

First, the effect of sodium tetraborate on the kinetics and equilibriumof isomerization for unaltered XI pellets in a buffer of pH 7.5 wasstudied. These data are shown in FIG. 3 curve A. When compared with thecorresponding data obtained in the absence of borate (FIG. 2 curve A),even at this low concentration (0.05M) borate is able to shift theequilibrium significantly in favor of higher xylulose production. Theequilibrium concentration of xylulose reaches ˜30 g/l, which is morethat 3 times that seen without borate (˜9 g/l). Borate addition leads toan increased conversion of xylose and a shift in the equilibriumxylose:xylulose ratio from ˜6:1 to ˜1:1.

Urease Co-Immobilized Enzyme Pellets:

The effect of urease immobilization on the pellets has a negligibleimpact on the overall kinetics and equilibrium achieved at pH 7.5 asshown in FIG. 3, curves A and C. (both run without urea). Theimmobilized urease may add a small mass transfer resistance, which couldaccount for the slowing of the kinetics seen as the xylose concentrationdecreases. Next, upon adding urea (0.01M) to the citrate buffersolution, there is a significant formation of xylulose with theco-immobilized enzyme pellets, with xylulose reaching a concentration of˜17 g/l by 48 hrs. This value is much higher than the correspondinglevel reached without borate addition, which was about 5 g/l (see FIG. 2curve B, and FIG. 3, curve B).

Referring again to the reactions given in Equations 1 and 2, theformation of tetrahydroxyborate ions is affected by the pH of themedium. Consequently, the ability of borax to shift the xylose:xyluloseisomerization equilibrium is also a function of pH. At low pH (4 to 5)very few tetrahydroxyborate ions are formed (as the second reaction doesnot occur) and accordingly borax is less likely to have any influence onthe isomerization equilibrium. On the other hand, in the higher pH range(6 to 8), the tetrahydroxyborate ion concentration reaches appreciablelevels, and these ions bind strongly to xylulose (Eq. 3), shifting theisomerization equilibrium.

As shown in FIG. 4, in the two-pH environment co-immobilized enzymepellet system, the core region of the pellets (where the pH is high andXI is active) provides conditions conducive to strong binding ofxylulose to tetrahydroxyborate ions and formation of the xylulose-boratecomplex (Xu-B). However, in the bulk solution where the pH is low, verylittle borate-sugar complex formation takes place. The likely net resultof this two-pH environment in the context of SIF is that boric aciddiffuses into the pellets, is converted to tetrahydroxyborate ions (Eq.2), binds to xylulose, and ferries xylulose from inside the pellet tothe bulk solution outside. In the low pH bulk solution, the Xu-Breleases xylulose and the borate ions recombine with hydrogen ions toform boric acid. Thus, tetrahydroxyborate, in addition to shifting theisomerization equilibrium, facilitates the removal of xylulose from thecore of the pellets into the bulk where xylulose can be readilymetabolized by yeast to ethanol. Xylose feed solutions isomerized in thepresence of 0.05M borate have been used in fermentation studies withyeast, and no inhibition of yeast by borate have been observed [45].

Effect of Co-Immobilized Enzyme Pellet Mass on Isomerization

The activity of the SWEETZYME™ pellets co-immobilized with ureasedepends on many factors. These factors include the concentration of ureaand the pH in the bulk solution and the activity of urease immobilizedin the outer layer (Zone 1) of the pellet. These factors influence theproduction of ammonia and the neutralization of the diffusing hydrogenions, and hence the size of the active XI zone (Zone 2).

In FIG. 5, transient xylulose production is shown as a function of totalco-immobilized enzyme pellet mass. All pellets used were from the sameco-immobilization batch and have the same urease and XI loadings.Experiments were conducted at 34° C. and pH 4.5 with 0.01M urea, 0.05Msodium tetraborate, and an initial xylose concentration of 60 g/l.Experiments shown in FIG. 5 curves B and C have 3.3 (18 g/l) and 6.6 (36g/l) times more of each enzyme compared to curve A (5.2 g/l). At timezero in all experiments, the interior pH increases rapidly to valuescloser to the optimum for XI activity as ammonia is produced. Inexperiments B and C, the increased mass of urease and XI will cause amore rapid decrease in the bulk urea and xylose concentrations than inA. As the bulk urea concentration decreases, the ammonia production perpellet decreases and the interior pH also starts to decrease. This dropin pH occurs earlier in cases where the total urease mass (activity) ishigher, leading to an accompanying loss in specific XI activity.

From the data shown in FIG. 5, the average specific XI activity wascalculated for the first hour of isomerization; these results aresummarized in Table 1 which shows the effect of co-immobilized enzymepellet mass on isomerization kinetics and xylulose production. Total XIis proportional to the pellet mass, but the XI activity measured overthe first hour depends on the internal pH profile within the pellet. Aspellet mass increases, the bulk urea concentration decreases morerapidly and the changing internal pH profile results in an apparentdecrease in specific XI activity. Although urea consumption and loss ofXI activity occurs most rapidly for the highest pellet mass, the totalxylulose produced while the XI is active is the greatest.

TABLE 1 Ave XI Specific XI Mass Of Activity In Specific Activity ExptPellets First hr In First hr (Ave) Final [Xylulose] A 0.13 g  7.8 U 58.5(U/G Pellet) 20.0 g/L B 0.45 g 20.4 U 45.3 (U/G Pellet) 34.4 g/L C  0.9g 34.9 U 38.8 (U/G Pellet) 44.0 g/L *1 u = 1 μmol of xylulose producedper minute at 34° C. and bulk pH of 4.5.

The average specific XI activity (based on xylulose production per g ofpellets at pH 4.5) decreases with increasing pellet mass. However, thecorresponding total XI activity is higher, resulting in a much morerapid production of xylulose and much higher xylulose yield by 48 hrs.

The xylulose concentration (˜44 g/l) at 48 hrs for the highest pelletmass is substantially higher than the value achieved (˜30 gl/) withunaltered pellets at pH 7.5 with the same borate concentration (FIG. 3curve A). For an unaltered SWEETZYME™ pellet, the kinetics of theisomerization depend on the XI activity, but the equilibrium is governedsolely by the thermodynamics and is unaffected by the XI activity andpellet mass.

In the co-immobilized enzyme pellet system, there are xyluloseconversions that are higher than those possible with the unalteredpellets at pH 7.5. As shown in FIG. 3 and FIG. 4, in the co-immobilizedenzyme pellet system, tetrahydroxyborate acts to shift the equilibriumby binding to xylulose and also shuttles complexed xylulose from thepellet interior to the bulk solution. While not wishing to be bound bytheory, the inventors herein believe that this dual role oftetrahydroxyborate is responsible for the significant improvement inxylose conversion seen in the co-immobilized enzyme pellet system when apH gradient is established.

Effect of Urea:

The urea concentration in the bulk media will affect the rate andquantity of ammonia produced and, hence, the maintenance of the pHgradient within the co-immobilized enzyme pellet. The urea concentrationwill also determine the volume of the active XI core and this will, inturn, influence the kinetics of the isomerization and the extent ofisomerization. In FIG. 6 transient xylulose production is shown as afunction of urea concentration. All pellets used were from the sameco-immobilization batch and have the same urease and XI loadings.Experiments were conducted at 34° C. and pH 4.5 with either 0.01M (FIG.6, curve A) or 0.1M urea (FIG. 6, curve B), 0.05M sodium tetraborate,and an initial xylose concentration of 60 g/l.

As seen in these two experiments, the rate of xylose isomerization isvery similar for the first 4 hours. For both cases, concentration ofurea is significantly higher than the K_(m) for urease so the internalpH profiles within the pellet are likely to be similar. Since thepellets also have the same XI loading, xylulose production is equivalentin both. However, by 8 hrs, urea consumption in FIG. 6, curve A, resultsin a decrease in reaction velocity for urea hydrolysis. With reducedammonia production, the internal volume of the pellet with active XIdecreases, and a drop in xylulose production relative to FIG. 6, curveB, is observed. Based on the results shown for FIG. 6, curve A, ureahydrolysis is no longer effective at maintaining the two pHmicroenvironments by 24 hrs. For FIG. 6, curve B, with a much higherinitial urea concentration, the active zone for xylose isomerization ismaintained for a much longer period of time (>48 hrs). The finalxylulose concentration measured at 48 hrs was ˜52 g/l, corresponding toa xylose:xylulose ratio of ˜1:6.5.

Effect of Co-Immobilized Enzyme Pellet Mass on Isomerization in Presenceof Excess Urea:

The effects of pellet mass and urea concentration on the finalcomposition of the isomerization solution are summarized in FIG. 7.Pellet mass ranged from 0.13 g to 0.9 g per experiment, while theinitial urea concentrations were either 0.01 or 0.1M. For 0.01M urea(FIG. 7, B and C), the increase in pellet mass results in an increase inthe rate of xylulose production (see also FIG. 5) as well as an increasein the total xylulose produced. However, none of the experiments with0.01M urea reach a xylulose yield as high as that achieved with thelowest pellet mass when 0.1M urea is added. For 0.1M urea (FIG. 7, D andE), the increase in pellet mass also results in an increase in the rateof xylulose production and a reduction in the time required to reach thefinal solution composition, but the final xylulose yields remainunchanged. Although increasing the pellet mass (more XI) increases theisomerization kinetics, urea plays an essential role in maintaining XIactivity and achieving high xylulose yields. The co-immobilized enzymesystem, by virtue of the unique two-pH microenvironments and the borateshuttling of xylulose to the bulk, results in conversion of xylose toxylulose (˜86%) that is significantly higher than that achievable withthe native XI at its optimal pH (FIG. 7,F).

Effect of Metal Ion Addition on Xylose Isomerization

In addition to evaluating the effectiveness of borate in favorablyshifting the xylose to xylulose equilibrium, maintaining sustainedoptimal activity of XI for long time periods was also considered. The XIenzyme requires metal ions for activity, and these ions can be depletedduring the isomerization. The inventors tested whether improvement inactivity of XI can be realized by the addition of Mg²⁺ and Co²⁺ ions tothe medium.

In these experiments, unaltered SWEETZYME™ at pH 7.5 was used. In theabsence of borate, addition of metal ions results in a small shift inthe isomerization toward xylulose (FIG. 8 curves A and B). In thepresence of borate, a similar shift in the isomerization is observed(FIG. 8 curves C and D). Thus, metal ions either alone or in conjunctionwith borate provide an incremental improvement in the xyluloseproduction, but the effect is not as significant as the shift associatedwith addition of sodium tetraborate. In certain embodiments,supplementing with metal ions may contribute to the long-term activityand reusability of the particles.

SIF of Poplar Hydrolysate with Isomerization Pellets Added to aFermentor

When unfiltered hydrolysates are used for fermentation, the HFMF cannotbe used due to plugging of the hollow fibers. SIF was conducted in threedifferent fermentations wherein the broth contained: (1) pure xylose,(2) equal proportions of glucose and xylose, and (3) poplar hydrolysate.Borate, which was used in all experiments, did not adversely affecteither the yeast viability or their ability to produce ethanol.

In simultaneous-isomerization-and-fermentation (SIF) experiments, theco-immobilized enzyme pellets were added to the broth in a standardfermentor, initial isomerization was allowed for 24 hrs, and then yeastwas added to initiate fermentation.

As shown in Table 2 for pure xylose, the inventors are able to fermentto 98% of the theoretical yield of ethanol in 28 hrs. To simulatehydrolysate, the inventors also fermented a sugar media containing equalamounts of glucose and xylose. These results for mixed sugars show thatin SIF mode, the inventors were able to ferment nearly all of theavailable glucose and xylose and produce 88% ethanol yield in only 36hrs. For the poplar hydrolysate, complete conversion of xylose is seenat the end of fermentation, indicating that SIF occurs.

TABLE 2 Yield^(a) % T Time Concentration (g/l) g EtOH/ EtOH Yeast pH (°C.) (hr) Glucose Xylose Xylulose EtOH g sugar yield C. shehatae 5.5 3048 — — 50 17.5 0.350 68.6%  NJ 23^(b) ATCC 2358^(c) 5 35 56 50 50 — 390.390 76.5%  Baker's yeast^(d) - 5 34 28 — 30 — 15 0.501 98% pure sugarBaker's yeast^(d) - 5 34 36 30 30 27 0.45 88% mixed sugars Baker'syeast^(d) - 5 34 24  9  4 6.5 0.50 98% IL-pretreated poplar hydrolysate^(a)The theoretical ethanol yield is 0.51 g ethanol per g sugar.^(b)Adapted strain [1]. ^(c)Unaltered XI for xylose to xyluloseisomerization at compromised pH [2]. ^(d)Data from our laboratory usingco-immobilized enzyme technology. The nutrients used are: 0.3% yeastextract, 0.6% peptone, 0.17% diammonium phosphate, 4.6 mM sodium azide,0.1M urea, 0.05M borate, and 18 g/l pellets.

Simultaneous-Isomerization and Fermentation (SIF) System Use with aFiltered Biomass Hydrolysate:

FIG. 9 is a schematic illustration of a packed bed of co-immobilizedenzyme pellets and a fermentor module configurations for an SIF process.The hollow fiber membrane fermentor (HFMF) shows a blow-up of amicroporous fiber and yeast cells. Sugar flows through fiber lumens andyeast grow in the space surrounding the fibers. Sugars diffuse throughpores in the fiber wall to yeast, where sugars are fermented to ethanol.Ethanol diffuses back through the pores into the fiber lumen and flowsout of the device at the end. The yeast cells are confined to the shellspace and can be loaded at a very high density per unit volume as shown.

This configuration provides a fermentation beer that is free from yeastand can be easily concentrated for ethanol recovery. After the ethanolhas been distilled off from the fermentation beer, the remaining aqueoussolution containing buffers and borate can be recycled to the upstreamprocess units (i.e. hydrolysis/isomerization), resulting in significantcost savings in consumables. This configuration provides a facile methodfor recovery and reuse of the isomerization catalyst pellets as they areconfined to the packed bed and do not come into direct contact withyeast. This configuration allows for a high density of yeast in the HFMFwhich is needed for xylulose fermentation and also allows extended useof the yeast for fermentation. Unlike traditional fermentors, the yeastis not disposed of after each batch of fermentation. The modular natureof this configuration allows for easy scale-up of the SIF processwithout significant capital costs.

Use of Modular Configuration for SIF.

A packed bed reactor containing 4.5 g of urease-coated GENSWEET™ pelletswas used for the isomerization of xylose. The reactor is a cylindricalcolumn 30 cm in length with a 1 cm inner diameter (Kontes Chemistry andLife Sciences Products). An F50NRe HEMOFLOW™ hollow fiber fermentor wasloaded with 50 g of dry yeast for the fermentation of the sugars toethanol. A volume of 250 ml of a nutrient buffer containing 60 g/lglucose and 30 g/l xylose was then circulated at a rate of 30 ml/min forthe isomerization and fermentation. Three different cases were run at34° C. and pH 4.5: 1) fermentation with no pellets in the packed bedunit (no isomerization); 2) 1 hr of pre-isomerization of the sugarsolution followed by simultaneous-isomerization-and-fermentation (SIF);and 3) simultaneous-isomerization-and-fermentation.

The results of these experiments are summarized in Table 3. For thethree cases, all glucose is converted to ethanol within 2 hrs.

TABLE 3 Ethanol Yield Yield Time Glucose Xylose Xylulose Ethanol (gEtOH/ (% of (Hr) (g/l) (g/l) (g/l) (g/l) g sugar theoretical) Case 1: Noisomerization 0 61.98 30.11 0.00 0.00 0.00  0% 2 0.00 17.38 0.00 29.540.40 78% 4 0.00 16.34 0.00 30.01 0.40 78% 24 0.00 14.41 0.00 30.93 0.4078% Case 2: SIF with 1 hr of pre-isomerization 0 62.97 15.01 15.64 0.000.00  0% 2 0.00 4.90 7.48 33.09 0.41 80% 4 0.00 4.09 4.95 35.59 0.42 83%24 0.00 1.76 0.52 41.22 0.45 88% Case 3: SIF 0 60.20 30.91 0.00 0.000.00  0% 2 0.00 18.08 3.05 31.82 0.45 89% 4 0.00 18.08 2.88 34.03 0.4995% 24 0.00 14.55 0.00 37.22 0.49 95%

In Case 1, although xylose is consumed, no ethanol is produced from thissugar as the ethanol concentration is constant between 2-24 hrs. In theabsence of the xylose isomerase (XI) no xylulose is formed; the xyloseis being utilized by yeast to form other metabolic products such asxylitol and arabitol.

For Case 2, half of the xylose is isomerized to xylulose prior to theinitiation of fermentation. By 24 hrs, all of the glucose, all of theinitial xylulose, and a fraction of the initial xylose have all beenconverted to ethanol. The presence of XI allows for rapid conversion ofxylose to xylulose and to ethanol which reduces the flux of xylose toother byproduct pathways. These data indicate that isomerization ofadditional xylose and fermentation of xylulose occurs during the 24 hrperiod with a corresponding higher ethanol yield than for Case 1.

For Case 3, the data show that xylulose is fermented rapidly uponproduction as beyond 5 hrs, the concentration of xylulose is zero.However, the concentration of ethanol continues to increase and hasreached 95% of the theoretical yield by 24 hrs.

Isomerization and Fermentation of a Poplar Hydrolysate in the ModularConfiguration

This example demonstrates the viability of the system described hereinwith a biomass hydrolysate. Although model sugar solutions contain themajor sugars present in hydrolysate, actual hydrolysates could containadditional hydrolysis products that can be inhibitory to fermentationand isomerization. To verify how the co-immobilized enzyme pellets aswell as the fermentor (HFMF) perform with actual hydrolysate, we haveused a 5% w/v poplar hydrolysate prepared with a proprietary ionicliquid pretreatment method. As such, the co-pending patent applicationSer. No. 11/710,357 and Ser. No. 12/075,762, are incorporated herein byreference in their entireties.

A 200 ml volume of hydrolysate was first isomerized in a packed bedcontaining XI pellets and then fermented in the HFMF. Isomerization wascarried out with 0.05M borate for 24 hours; 91% of the xylose wasisomerized to xylulose. Results of the fermentation are summarized inTable 4 which shows ethanol yield from poplar hydrolysate in an HFMF.

TABLE 4 Based on total Based on fermentable fermentable sugar* sugar*consumed g ethanol/g sugar 0.42 (83%) 0.50 (98%) *Fermentable sugar isthe sum of initial glucose and xylose before isomerization.

This combination of a packed bed of co-immobilized enzyme pellets and ahollow fiber fermentor module configurations for an SIF process hasseveral unique and unexpected advantages: (i) it provides a fermentationbeer that is free from yeast and can be easily concentrated for ethanolrecovery. (ii) After the ethanol has been distilled off from thefermentation beer, the remaining aqueous solution containing buffers andborate can be recycled to the upstream process units (i.e.,hydrolysis/isomerization), resulting in significant cost savings inconsumables. (iii) This configuration provides a facile method forrecovery and reuse of the isomerization catalyst pellets as they areconfined to the packed bed and do not come into direct contact withyeast. (iv) This configuration allows for a high density of yeast in theHFMF which is needed for xylulose fermentation and also allows extendeduse of the yeast for fermentation. (v) Unlike traditional fermentors,the yeast is not disposed of after each batch of fermentation. (vi) Themodular nature of this configuration allows for easy scale-up of the SIFprocess without significant capital costs.

Modular SIF Configuration for Use with Filtered Hydrolysates.

The production of cellulosic ethanol is impacted by the ability torecover lignin. In certain embodiments, it may be necessary to filtratethe hydrolysate prior to fermentation.

In another aspect, there is provided herein a modular SIF configurationthat is capable of using a clarified hydrolysate. FIG. 9 shows aschematic illustration of a packed bed system 10. The modular SIFconfiguration allows for the easy and rapid reuse of the co-immobilizedenzymes and for a flexible and efficient fermentor operation.

The packed bed system 10 includes a batch vessel 12, a packed bed column14 having a inlet 15, an outlet 16, and containing a desired quantity ofco-immobilized enzyme pellets 18, and a fermentor 20, having an inlet 21and an outlet 22. In certain embodiments, the fermentor 20 comprises amicroporous, hollow-fiber membrane 24 that separates yeast 26 from flowchannels 28 in the fermentor 20.

Isomerization of xylose is conducted in the packed bed column 14containing the immobilized enzyme pellets 18, while fermentation isconducted in the fermentor 20. The batch vessel 12, the packed bedcolumn 14 and the fermentor 20 are connected in a closed loop systemsuch that glucose and xylose flow into the inlet 15 of the packed bedcolumn.

In operation, the glucose and xylose from the batch vessel 12 come intocontact with the co-immobilized enzyme pellets 18 where theisomerization reaction described herein occurs. Then glucose andxylulose flow from the packed bed outlet 16 and into the fermentor inlet21. The glucose and xylulose sugars pass through the membrane and comeinto contact with the yeast 26. As the yeast uses these sugars, ethanolis produced. The ethanol then passes through the membrane into the flowchannel 28, and exits through the fermentor outlet 22.

By connecting the packed bed system 10 in a series configuration and byoperating in a recycle mode, a “simultaneous isomerization+fermentation”SIF operation is achieved. The packed bed system 10 allows for repeateduse of the pellets 18 and the fermentor 20 for a large number of batchfermentations. The packed bed system confines the yeast 26 to a shellside of the hollow fiber membrane fermentor 20 (HFMF), avoiding directcontact between the yeast and the immobilized enzyme pellets, whichmakes the recovery and reuse of the pellets very easy. Furthermore, incertain embodiments, the fermentor 20 has yeast densities high enough topack the shell side such that further growth of the yeast is preventedand sugars are rapidly fermented to ethanol. The packed bed system 10 isparticularly advantageous for xylulose utilization.

FIGS. 10 and 11 shows data for the two processes: [(1) packed bed, and(2) HFMF operated separately. For the packed bed isomerization, 0.9 g ofimmobilized enzyme pellets were placed in the bottom of a 5 ml glasssyringe. Using a peristaltic pump, 50 ml of media containing 30 g/lxylose, 0.05 M borate and 0.05 M urea was recirculated from a reservoirthrough the bed at 1 ml/min while maintaining a 1 cm liquid head abovethe enzyme pellets in the column. The packed bed set-up was assembled inan incubator to maintain a constant temperature of 34° C. Samples werecollected from the reservoir for HPLC analysis of xylose and xylulose. Acontrol experiment with the pellets from the same immobilization batchwas run in an agitated shake flask. The transient xylulose concentrationand yield for the packed bed and shake flask experiments are as shown inFIG. 10. Both the rate of formation and overall yield of xylulose arehigher in the packed bed, indicating that the time for isomerization maybe practically reduced from ˜24 hrs to less than 10 hrs due to the moreefficient mass transfer of sugars to the enzymes in the packed beddesign.

For the fermentation unit, a F50NR HEMOFLOW™ cartridge (FreseniusMedical Care North America) was used. The microporous fibers are made ofpolysulfone with a molecular weight cut-off of 25 kDa, and totalmembrane surface area is 1 m². A port on the shell side of the cartridgewas used to introduce the yeast and to vent CO₂ produced during thefermentation. Yeast (Sigma YSC2-500G, 25 g) were inoculated and culturedon the shell-side (150 ml volume) of the fibers at 34° C. 250 ml ofmedia containing 60 g/l glucose was pumped through the fiber lumens (50ml volume) at flow rates ranging from 1-70 ml/min to determine theeffect of flow rate on the ethanol fermentation rate. The concentrationof ethanol was measured through a sample port off of the batch vessel.

As shown from FIG. 11, glucose was consumed within 6 hours at all flowrates, which is comparable to our batch fermentation results (data notshown). For the 30-50 ml/min range, glucose was consumed even fasterthan in our batch experiments, indicating that the HFMF may have atime-advantage over batch fermentation. The ethanol yield was nearly100% for all runs, indicating minimal by-product production. These timescales are comparable to those of isomerization in the packed column,thus providing another significant advantage over the art.

It is to be understood that other sugar solutions and hydrolysates canbe used to establish module sizing to minimize SIF time. Also, themodules can be scaled to accomplish larger-scale SIF. It is also to beunderstood that benchmarks for reusability and lifetimes for the modulescan be readily established.

System for Easy and Rapid Reuse of the Co-Immobilized Enzymes for Usewith Solids-Containing Hydrolysates

If the pellets are added directly to a fermentation vessel withsolids-containing hydrolysate, their recovery is extremely difficult.Moreover, simultaneous saccharification and fermentation (SSF) ofbiomass, which has several process advantages, necessitates the handlingof solids-containing fermentation media.

To address the recovery and reuse of the pellets (which are a very smallfraction of the residual solids) in these situations, there is alsoprovided herein a system that allows for the easy and rapid reuse of theco-immobilized enzymes for use with solids-containing hydrolysates.

In one non-limiting example, for larger scale batch fermentations, theco-immobilized enzyme pellets can secured in a confinement system. Onexample of a suitable confinement system is a spinning basket system 30,as schematically illustrated in FIG. 12.

In this embodiment, the enzyme pellets 32 are packed in flat baskets 34that form the vanes 36 of an impellor 38 that is rotated in thefermentation broth. By varying the selection of materials for the basketand by optimizing rotation speeds, attachment of solids from thefermentation broth to the baskets can be minimized. In addition, CO₂generated during the fermentation will score the surfaces of thebaskets, further preventing material build-up. One advantage of thespinning basket system 30 is that this confinement method is especiallyuseful in large-scale ups in conducting heterogeneous chemicalreactions.

FIG. 13: Examples of process configurations using co-immobilized enzymetechnology. Process A—Using co-immobilized enzyme pellets withcellulases at pH 4.8 and 50° C. in the presence of borate and urea canenable simultaneous saccharification and xylose isomerization (SSI). Theresulting sugar mix of glucose and xylulose can be fermented by nativeyeasts. Process B—Adding co-immobilized enzyme pellets and native yeastto biomass hydrolysate at pH 4.5 and 35° C. in the presence of borateand urea allows simultaneous isomerization and fermentation (SIF) withclose to theoretical ethanol yields from all biomass sugars. ProcessC—Simultaneous saccharification and fermentation (SSIF) of C6 and C5sugars using native yeasts can be done with co-immobilized enzymepellets in a medium maintained at pH 4.8 and 35° C. containing borate,urea, cellulases, and native yeast.

As shown in FIG. 13, cellulosic ethanol can be produced in a variety ofoperating configurations. Non-limiting examples include, in particular,co-immobilized enzyme pellets enable the performance of:

Process A—simultaneous saccharification and isomerization (SSI);

Process B—simultaneous isomerization and fermentation (SIF), and

Process C—simultaneous saccharification-isomerization and fermentation(SSIF).

In certain embodiments, both the enzymes XI and urease have significantactivity in the temperature range of 35-70° C. Indeed, their optimalactivity is closer to 50° C., just as for the cellulases. However,unlike cellulases which have optimal activity at pH 4.8, the pH optimumfor XI is 7.5. Since the methods described herein can overcome this pHdiscrepancy by production of the two pH microenvironments, Process A“SSI” can be conducted at elevated temperature, significantly enhancingthe kinetics of isomerization and the reaction equilibrium. This resultsin a significant reduction in the time needed for saccharification andfermentation. Further, when the Process A includes added borate and ureato the saccharification mileau of pretreated biomass, cellulase enzymeactivities are not adversely affected by these additives.

In addition, Process A (“SSI”) can be implemented with a confinementsystem which can enable the easy recovery of the enzyme pelletsfollowing saccharification. Following Process A (“SSI”), fermentation ofglucose and xylulose to ethanol can be performed in the same vessel bysimply reducing the hydrolysate temperature and adding native yeast.

It is to be noted that, in certain embodiments, Process B(“SIF”) isespecially suited to the use of bilayered pellets.

Also, in certain embodiments, with native yeasts, only the C6 sugars canbe fermented to ethanol in a simultaneous-saccaraification andfermentation mode (“SSF”), while the xylose portion remains unfermented.The inability of native yeasts to ferment xylose results in a loss ofnearly 40% of the available sugars from the biomass hydrolysate. Thus,the “SSF” mode may, in certain embodiments, be viable using only GMOsthat can ferment both the C6 and C5 sugars of biomass at a pH of ˜4.8.

As seen in FIG. 13, however, a unique feature of the co-immobilizedenzyme technology is that it allows isomerization and fermentation totake place at a pH of 4.5, a pH that is also typical of enzymaticsaccharification. This affords the opportunity to combinesaccharification, isomerization, and fermentation all into one step. Theconcentration of enzyme pellets is low relative to other solids ofbiomass, and no significant loss of cellulases by adsorption on theparticles would be expected. Also, in certain embodiments, the additionof bovine serum albumin (BSA) to the reaction medium can be done as ameans of preventing loss of cellulases due to non-specific adsorption onsolids other than polysaccharides.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to the particular embodimentdisclosed herein contemplated for carrying out this invention, but thatthe invention will include all embodiments falling within the scope ofthe claims.

REFERENCES

The references discussed above and the following references, to theextent that they provide exemplary procedural or other detailssupplementary to those set forth herein, are specifically incorporatedherein by reference. Citation of a reference herein shall not beconstrued as an admission that such is prior art to the presentinvention.

-   1. Zaldivar, J., Nielsen, J., and Olsson, L., Fuel ethanol    production from lignocellulose: a challenge for metabolic    engineering and process integration. Appl Microbiol Biotechnol., 56    (1-2) 2001 17-34.-   2. Office of Science, U.S. D. O. E., Breaking the Biological    Barriers to Cellulosic Ethanol: A Resarch Roadmap Resulting from the    Biomass to Biofuels Workshop. December 2005, U.S Department of    Energy: Rockville, Md.-   3. Holtzapple, M. T., Chapters, cellulose, hemicelluloses, and    lignin., in Encyclopedia of Food Science, Food Technology and    Nutrition., M. J. Sadler, Editor. 1993, Academic Press: London. p.    758-767, 2324-2334, 2731-2738.-   4. Somerville, C., Bauer, J., G., B., Facette, M., Hamann, T.,    Milne, J., Osborne, E., Paredez, A., Persson, S., Raab, T., Vorwerk,    S., and Youngs, H., Toward a Systems Approach to Understanding Plant    Cell Walls. Science, 306 (5705) 2004 2206-2211.-   5. Van Maris, A. J. A., Abbott, D. A., Bellissimi, E., Van Den    Brink, J., Kuyper, M., Luttik, M. A. H., Wisselink, H.,    Scheffers, A. W., Van Dijken, J. P., and Pronk, J. T., Alcoholic    fermentation of carbon sources in biomass hydrolysates by    Saccharomyces cerevisiae: current status. Antonie van Leeuwenhoek,    90 (4) 2006 391-418.-   6. Prior, B. A., Kilian, S. G., and Dupreez, J. C., Fermentation of    d-xylose by the yeasts Candida shehatae and Pichia stipitis, in    Process Biochem. 1989. p. 21-32.-   7. Tantirungkij, M., Nakashima, N., Seki, T., and Yoshida, T.,    Construction of xylose-assimilating Saccharomyces cerevisiae.    Journal of Fermentation and Bioengineering, 75 (2) 1993 83-8.-   8. Wang, P. Y., Shopsis, C., and Schneider, H., Fermentation of a    pentose by a yeasts. Biochem. Biophysics. Res. Communications, 94    1980 248-254.-   9. Chiang, L. C., Gong, C. S., Chen, L. F., and Tsao, G. T.,    D-Xylulose Fermentation to Ethanol by Saccharomyces cerevisiae.    Applied and Environmental Microbiology, 42 (2) 1981 284-289.-   10. Hsaio, H. Y., Chiang, L. C., Ueng, P. P., and Tsao, G. T.,    Sequential Utilization of Mixed Monosaccharides by Yeasts. Applied    and Environmental Microbiology, 43 (4) 1982 840-845.-   11. Gong, C. S., Chen, L. F., Flickinger, M. C., Chiang, L. C., and    Tsao, G. T., Production of Ethanol from D-xylose by using D-Xylose    Isomerase and Yeasts. Applied and Environmental Microbiology, 41 (2)    1981 430-436.-   12. Yu, S., Jeppsson, H., and Hahn-Gaegerdal, B., Xylulose    fermentation by Saccharomyces cerevisiae and xylose fermenting yeast    strains. Applied Microbiology and Biotechnology, 44 1995 314-320.-   13. Sarthy, A. V., Mcconaughy, B. L., Lobo, Z., Sundstrom, J. A.,    Furlong, C. E., and Hall, B. D., Expression of the Escherichia coli    xylose isomerase gene in Saccharomyces cerevisiae. Applied and    environmental microbiology, 53 (9) 1987 1996-2000.-   14. Kotter, P., Amore, R., Hollenberg, C. P., and Ciriacy, M.,    Isolation and characterization of the Pichia stipitis xylitol    dehydrogenase gene, XYL2, and construction of a xylose-utilizing    Saccharomyces cerevisiae transformant. Current genetics, 18 (6) 1990    493-500.-   15. Amore, R., Kotter, P., Kuster, C., Ciriacy, M., and    Hollenberg, C. P., Cloning and Expression in Saccharomyces    cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene    (XYL1) from the xylose assimilating yeast Pichia stipitis. Gene, 109    1991 89-97-   16. Moes, C. J., Pretorius, I. S., and Van Zyl, W. H., Cloning and    expression of the Clostridium thermosulfurogenes D-xylose isomerase    gene (xylA) in Saccharomyces cerevisiae. Biotechnology Letters,    18 (3) 1996 269-74.-   17. Walfridsson, M., Bao, X., Anderlund, M., Lilius, G., Buelow, L.,    and Hahn-Gaegerdal, B., Ethanolic fermentation of xylose with    Saccharomyces cerevisiae harboring the Thermus thermophilus xylA    gene, which expresses an active xylose (glucose) isomerase. Applied    and Environmental Microbiology, 62 (12) 1996 4648-4651.-   18. Hahn-Hagerdal, B., Wahlbom, C. F., Gardonyi, M., Van Zyl, W. H.,    Otero, R. R. C., and Jonsson, L. J., Metabolic engineering of    Saccharomyces cerevisiae for xylose utilization. Advances in    Biochemical Engineering/Biotechnology, 73 (Metabolic Engineering)    2001 53-84.-   19. Jeppsson, M., Johansson, B., Hahn-Gaegerdal, B., and    Gorwa-Grauslund, M. F., Reduced Oxidative Pathway flux in    recombinant xylose-utilizing strains improves the ethanol yield from    xylose. Applied and Environmental Microbiology, 69 2002 5892-5897.-   20. Johansson, B. and Hahn-Gaegerdal, B., The non-oxidative pentose    phosphate pathway controls the fermentation rate of xylulose, but    not of xylose in Saccharomyces cerevisiae. FEMS Yeast Research, 2    2002 277-282.-   21. Verho, R., Londesborough, J., Penttilae, M., and Richard, P.,    Engineering redox cofactor regeneration for improved pentose    fermentation in Saccharomyces cerevisiae. Applied and Environmental    Microbiology, 69 (10) 2003 5892-5897.-   22. Gardonyi, M. and Hahn-Hagerdal, B., The Streptomyces rubiginosus    xylose isomerase is misfolded when expressed in Saccharomyces    cerevisiae. Enzyme and Microbial Technology, 32 (2) 2003 252-259.-   23. Kuyper, M., Harhangi, H. R., Stave, A. K., Winkler, A. A.,    Jetten, M. S. M., De Laat, W. T. A. M., Den Ridder, J. J. J., Op Den    Camp, H. J. M., Van Dijken, J. P., and Pronk, J. T., High-level    functional expression of a fungal xylose isomerase: the key to    efficient ethanolic fermentation of xylose by Saccharomyces    cerevisiae? FEMS Yeast Research, 4 (1) 2003 69-78.-   24. Kuyper, M., Winkler, A. A., Van Dijken, J. P., and Pronk, J. T.,    Minimal metabolic engineering of Saccharomyces cerevisiae for    efficient anaerobic xylose fermentation: a proof of principle. FEMS    Yeast Research, 4 2004 655-664.-   25. Kuyper, M., Toirkens, M. J., Diderich, J. A., Winkler, A. A.,    Van Dijken, J. P., and Pronk, J. T., Evolutionary Engineering of    mixed-sugar utilization by a xylose-fermenting Saccharomyces    cerevisiae strain. FEMS Yeast Research, 5 2005 925-934.-   26. Dien, B. S., Cotta, M. A., and Jeffries, T. W., Bacteria    engineered for fuel ethanol production: current status. Applied    Microbiology and Biotechnology, 63 2003 258-266.-   27. Jeffries, T. W., Engineering yeasts for xylose metabolism.    Current Opinion in Biotechnology, 17 2006 320-326.-   28. Linden, T. and Hahn-Gaegerdal, B., Fermentation of    lignocellulose hydrolysates with yeasts and xylose isomerase. Enzyme    and Microbial Technology, 11 1989 583-589.-   29. Byers, J. P., Fournier, R. L., and Varanasi, S., A Feasibility    Analysis of a Novel Approach For the Conversion of Xylose to    Ethanol. Chem. Eng. Comm, 112 1992 165-187.-   30. Chandrakant, P. and Bisaria, V. S., Simultaneous bioconversion    of glucose and xylose to ethanol by Saccharomyces cerevisiae in the    presence of xylose isomerase. Applied Microbiology and    Biotechnology, 53 2000 301-309.-   31. Bhosale, S. H., Rao, M. B., and Deshpande, V. V., Molecular and    industrial aspects of glucose isomerase. Microbiological Reviews,    60 (2) 1996 280-300.-   32. Mitsuhashi, S, and Lampen, J. O., Conversion of D-Xylulose to    D-Xylose in Extracts of Lactobacillus Pentosus. Journal of    Biological Chemistry, 204 1953 1011-1018.-   33. Hochester, R. M. and Watson, R. W., Enzymatic Isomerization of    D-Xylose to D-xylulose. Archives of Biochemistry and Biophysics, 48    1954 120-129.-   34. Tewari, Y. B., Steckler, D. K., and Goldberg, R. N.,    Thermodynamics of the Conversion of Aqueous Xylose to Xylulose.    Biophysical Chemistry, 22 1985 181-185.-   35. Byers, J. P., Shah, M. B., Fournier, R. L., and Varanasi, S.,    Generation of pH Gradient in an Immobilized Enzyme System.    Biotechnology and Bioengineering, 42 1993 410-429.-   36. Fournier, R. L., Byers, J. P., Varanasi, S., and Chen, G.,    Demonstration of pH Control in a Commercial Immobilized Glucose    Isomerase. Biotechnology and Bioengineering, 52 1996 718-722.-   37. Boeseken, J., The use of boric acid for the determination of the    configuration of carbohydrates. Adv Carbohydr Chem, 4 1949 189.-   38. Foster, A. B., Zone electrophoresis of carbohydrates. Adv    Carbohydr Chem, 12 1957 81.-   39. Mendicino, J. F., Effect of Borate on the Alkali-catalyzed    Isomerization of Sugars. Journal of the American Chemical Society,    82 (18) 1960 4975-4979.-   40. Hsaio, H. Y., Chiang, L. C., Chen, L. F., and Tsao, G. T.,    Effects of borate on isomerization and yeast fermentation of high    xylulose solution and acid hydrolysate of hemicellulose. Enzyme and    Microbial Technology, 4 1982 25-31.-   41. Allen, K. N., Lavie, A., Glassfeld, A., Tanada, T. N., Jenny, D.    P., Carlson, S.C., Farber, G. K., Petsko, G. A., and Ringe, D., Role    of the Divalent Metal Ion in Sugar Binding, Ring Opening and    Isomerization by D-Xylose Isomerase; Replacement of a catalytic    metal by an amino acid. Biochemistry, 33 1994 1488-1494.-   42. Liu, H. H. and Shi, Y., The Reaction Pathway of the    Isomerization of D-Xylose Catalysed by the Enzyme D-Xylose    Isomerase:A theoretical study. Proteins: Structure, Function and    Genetics, 27 1997 545-55.-   43. Worthington, C. E., Enzymatic Assay of Urease from Jack Beans    (E.C.3.5.1.5), in Worthington Enzyme Manual. 1972, Worthington    Biochemical Corporation: Freehold, N.J. p. 146-148.-   44. Rizzi, G. P., On the Effect of Tetraborate Ions in the    Generation of Colored Products in Thermally Processed    Glycine-Carbohydrate Solutions. Journal of Agricultural and Food    Chemistry, 55 2007 2016-2019.-   45. Hsiao, H. Y., Chiang, L. C., Chen, L. F., and Tsao, G. T.,    Effects of borate on isomerization and yeast fermentation of high    xylulose solution and acid hydrolysate of hemicellulose. Enzyme and    Microbial Technology, 4 1982 25-31.-   46. Pastinen, O., Visuri, K., Schoemaker, H. E., and Leisola, M.,    Novel reactions of xylose isomerase from Streptomyces rubiginosus.    Enzyme and Microbial Technology, 25 1999 695-700.-   47. Callens, M., Kersters-Hilderson, H., Van Opstal, O., and De    Bruyne, C. K., Catalytic properties of D-xylose isomerase from    Streptomyces violaceoruber. Enzyme and Microbial Technology, 8 (11)    1988 696-700.-   48. Callens, M. H., Tomme, P., Kesters-Hilderson, W., Cornelis, R.,    Vangrysperre, W., and Debruyne, C. K., Metal ion binding to D-xylose    isomerase from Streptomyces violaceoniger., Biochemistry Journal,    250 1988 285-290.-   49. Gong et al. U.S. Pat. No. 4,490,468.

What is claimed is:
 1. A packed bed system comprising co-immobilized enzyme particles connected in series to a porous hollow fiber membrane fermentor, the co-immobilized enzyme particles comprising a first region having a first enzymatic activity and a second region having a second enzymatic activity.
 2. The system of claim 1, further comprising a fermentation beer substantially free from yeast, wherein the fermentation beer is capable of being concentrated for ethanol recovery.
 3. The system of claim 1, comprising a recyclable aqueous solution containing buffers and borate.
 4. The system of claim 1, comprising isomerization catalyst particles confined to a packed bed, wherein the particles do not come into direct contact with yeast.
 5. The system of claim 1, comprising a high density of yeast in the fermentor.
 6. The system of claim 5, wherein the yeast is reusable following fermentation.
 7. The system of claim 1, further comprising a batch vessel containing a mixture of glucose and xylose.
 8. The system of claim 7, further comprising: a column having a flow channel, the column containing a desired quantity of the co-immobilized enzyme pellets; wherein the batch vessel, the column, and the fermentor are connected in a closed loop system.
 9. The system of claim 7, wherein isomerization of xylose into xylulose occurs when the mixture comes into contact with the co-immobilized enzyme pellets.
 10. The system of claim 1, wherein the co-immobilized enzyme pellets comprise urease and xylose isomerase.
 11. A borate-enhanced system for the isomerization of xylose in a co-immobilized enzyme pellet having a two-pH environment comprising: a liquid mixture having an acidic pH; a first region having a first enzymatic activity and a non-acidic pH; and a second region having a second enzymatic activity.
 12. The system of claim 11, wherein the mixture includes borax.
 13. The system of claim 12, wherein the two-pH environment allows for a positive shift in equilibrium that results from a selective complexation of xylulose to tetrahydroxyborate ions formed from borax.
 14. The system of claim 11, wherein the two-pH environment allows for a substantially simultaneous isomerization and fermentation (SIF) step capable of sustaining two different pH-microenvironments in a single vessel, wherein the first pH is substantially optimal for xylose isomerization, and the second pH is substantially optimal for fermentation of xylulose.
 15. The system of claim 14, wherein the simultaneous isomerization and fermentation step further includes co-immobilization of urease with xylose isomerase.
 16. A borate-enhanced system for the isomerization of xylose in a co-immobilized enzyme pellet having a two-pH environment, the system comprising co-immobilized enzyme particles packed in a confinement system that is rotated in a fermentation broth, the co-immobilized enzyme pellets comprising a first region having a first enzymatic activity and a second region having a second enzymatic activity.
 17. The system of claim 16, configured to carry out one or more processes substantially simultaneously.
 18. The system of claim 17, wherein the processes include one or more of: a simultaneous saccharification and xylose isomerization (SSI) process; a simultaneous isomerization and fermentation (SIF); and a simultaneous saccharification and fermentation (SSIF).
 19. The system of claim 16, wherein the particles are bilayer particles.
 20. The system of claim 19, wherein the particles comprise urease and xylose isomerase. 